Polychlorinated biphenyls (PCB) immunoassay method, its components and a kit for use in performing the same

ABSTRACT

An immunoassay method that integrates a sample processing component that enables the testing of a variety of environmental matrixes and the components for performing the method, wherein the immunoassay utilizes a monoclonal anti-PCB antibody to detect the presence or absence of PCB based contamination in a sample when tested in a field or laboratory location.

This is a continuation of application Ser. No. 08/068,093 filed on May28, 1993, abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to methods, compositions and kits forperforming PCB immunoassays.

2. Discussion of the Background

Testing is an essential, and integral, component of all environmentalprotection and restoration activities. It is the rate limiting elementthat influences the time, cost, and overall efficiency of projectmanagement.

The management of toxic waste sites usually involves a progressionthrough the stages of identification, characterization, remediation andmonitoring, with testing being performed during each phase. Referencelaboratory methods can effectively identify and quantify unknowncompounds in a sample, but become relatively inefficient when used torapidly locate contamination (i.e., mapping), and assist in remediationand monitoring activities. The complexity of laboratory protocols, andthe proximity of the labs to the test site, delays the availability ofinformation and increases the cost of data. The ultimate cost is in thetime required by the field crews. Effective field screening methods canincrease the efficiency of the clean-up process by providing an on-site,high-throughput, and cost-effective way to locate contamination andmanage its remediation.

The Environmental Protection Agency (EPA) has long promoted andsupported the concept of screening methods to supplement laboratoryanalysis and increase overall efficiency. The need for more effectivemethods has been recognized in the Superfund Amendments andReauthorization Act of 1986 which specifies the development andevaluation of alternative time and cost-saving methods that will assistin the eventual remediation of the nations Superfund sites.

Effective field screening methods can increase the efficiency of sitemanagement and improve overall data quality when used to supplement theservices of regional laboratories. The development of these methods,however, requires a technology that will be compatible with numerouscompounds and matrixes and yet be simple, effective and rugged enough tobe incorporated into a protocol for use in the field.

Screening methods need to provide fast, simple, cost-effective andreliable information when operated under field conditions. The reagentsand equipment should be portable and stable at ambient conditions, andthe claims relating to performance should accurately reflect anticipatedfield use. The methods should be able to rapidly provide an amplequantity of data, and the protocol should be simple to perform and safeto use. Performance characteristics relative to sensitivity, freedomfrom matrix interferences and cross-reacting compounds, and correlationto an acceptable reference method should be carefully evaluated.Developers must maintain high, and consistent, quality standardsrelative to the consistency of their manufacturing protocols, theadequacy of in-process and pre-release quality control methods, and thereliability of their product claims. A necessary characteristic ofparticular significance for screening methods, is that they exhibit avery low frequency of false negative results.

Screening methods detect contamination at specified concentrations. Theconcentration may relate to a hazardous threshold, a clean-up target, ora process-control parameter. The potential implications of falsenegative data far outweigh those of false positive results. Theconsequence of a false positive, while a costly problem that needs to beminimized, results in additional testing or treatment. False negativedata, however, provides an erroneous perception of a clean site, and mayhave serious environmental and legal consequences. Safeguards thatminimize the incidence of false negative results are imperative.Appropriate control over the frequency of false positive data needs tobe established and maintained.

The field of immunochemistry,, and the development of immunoassaytechnology, has been evolving since the late 19th century. However, themajority of these methods have been developed for use by the medicalcommunity. These methods have achieved a reputation for reliability andcost-effectiveness. Literally hundreds of immunoassays have beendeveloped for such applications as drug testing, Therapeutic DrugMonitoring (e.g. digitalis derivatives, anti-asthma formulations,anti-epileptic regents, antibiotics), pregnancy testing, hormone testing(e.g., thyroxine, thyroid stimulating hormone), tests for pathologicalmarkers (e.g. lactic dehydrogenase isozymes, creatine kinase isozymes),tests for acute phase proteins (e.g., carcinoembryonic antigen, alphafetoprotein) and tests for tumor marker proteins.

Environmental applications have been explored for the better part of adecade and a number of immunoassay methods have been developed. Mosthave been used for the detection of herbicides and pesticides in aqueousmatrixes. The application of immunoassay technology to the testing ofsolid waste, complex matrixes, and highly lipophilic compounds, hasprovided unique challenges for the chemist. The feasibility ofdeveloping such methods, however, has been demonstrated withimmunoassays for single compounds such as Dioxin (see, for example,Vanderlaan et al, Environmental Toxicology and Chemistry, 7:859-870,1988; and Stanker et al, Toxicology, 45:229-243, 1987).

The history of immunoassay technology can be traced to 1900 when KarlLandsteiner described the A, B and Zero (0) blood types after observingthe agglutination reaction (i.e., aggregation) that resulted when hemixed the erythrocytes and serum for several of his co-workers on aslide. His observation became the basis for present day blood typingmethods. Landsteiner remained a dominant figure in immunology for thenext 40 years performing numerous experiments that demonstrated theextraordinary specificity of the antibody binding reaction. Heintroduced the term "hapten" to define compounds that are unable todirectly stimulate antibody production when injected into an animal, butare capable of binding to an antibody if they are produced by analternate means. Most environmental chemicals are haptens, and althoughpotentially toxic, will not stimulate the immune system to respond.

For 50 years following Landsteiner's discovery, immunoassay technologycontinued to rely upon the binding and cross-linking ability of anantibody to cause agglutination, cell lysis, and protein flocculationreactions. These methods were relatively insensitive when compared tothe immunoassay methods of today, and better suited to the analysis oflarger compounds and organisms (e.g. bacteria, proteins). A majoradvance occurred in the 1950's when Drs. Berson and Yalow, whileinvestigating the metabolism of radio-labelled insulin administered todiabetic patients, observed the production of anti-insulin antibodies inthe serum of these patients (see Principles of Competitive ProteinBinding Assays, Second Ed., Odell, W. D. and Franchismont, P. (Eds.);Wiley and Sons, New York). They described a radioimmunoassay (RIA)method in 1959 that used anti-insulin antibody molecules andradio-labelled insulin in a highly sensitive procedure to quantifyinsulin levels in the serum. The RIA method used a competitive antibodybinding reaction, where radio-labelled insulin and sample insulincompete for a limited number of antibody binding sites. In 1977, RosalynYalow was awarded the Nobel Prize in Medicine for her work on thedevelopment of the radioimmunoassay method for peptide hormones (seeBasic and Clinical Immunology, 7th Ed. Stites, D. P. and Ten, A. I.editors; Appleton and Lange, Conn., 1991). RIA rapidly became auniversally accepted method that demonstrated exceptional specificity,sensitivity, and simplicity.

A simpler, safer, and more convenient immunoassay was reported in 1971,when two independent research teams, Engvall and Perlmann, and VanWeeman and Shuurs, simultaneously disclosed a competitive immunoassaymethod that used an enzyme-labelled conjugate instead of aradio-labelled-conjugate to produce a test that generated a visibleend-point signal (see Engvall et al, Immunochem. 8:871-874, 1971 and VanWeeman et al, FEBS Letters, 15:232-236, 1971). The new ELISA (i.e.,enzyme linked immunosorbent assay) method eliminated the problemsassociated with the safety, disposal and detection of radioactivereagents. The method offered long term stability, the opportunity togenerate quantifiable data using instruments commonly available in mostlaboratories, and a mechanism to develop separation-free (homogeneous)procedures and simple qualitative screening tests.

Current immunoassay technology benefits from the diversity of detectionsystems developed that use enzyme-catalyzed chromogenic reactions,radionuclides, chemiluminescence, fluorescence, fluorescencepolarization and a variety of potentiometric and optical biosensortechniques. Improvements in the sensitivity achieved has necessitatedthe generation of new descriptive nomenclature for methods that can nowdetect "zeptomolar" (10⁻²¹, 600 molecules) concentrations.

Immunoassay methods combine the specific binding characteristics of anantibody molecule with a read-out system that is used to detect andquantify compounds. Antibodies are binding proteins that are produced bythe immune system of vertebrates in response to substances that areperceived to be foreign.

The physiological role of antibody, or immunoglobulin, molecules is tobind, and thereby label for destruction, the perceived foreignsubstance. Antibody molecules are synthesized by a subset oflymphocytes, termed B lymphocytes, that become activated to produceantibody after exposure to substances having prerequisite size,complexity and "foreignness" to the host organism. Antibodies are large,polymeric proteins (i.e. ≧1.5×10⁵ d), that can be classified intosub-populations on the basis of their sequence, size and number ofsubunits. Five major populations, or isotypes, exist carrying thedesignations of IgM, IgA, IgD, IgG and IgE, with immunoglobulin G (IgG)usually found in the highest concentration.

Immunochemical assays are reliable when used in the screening of soilfor contamination and have been used commercially for the rapid analysisof a variety of compounds (see Nqo, in Enzyme-Mediated Immunoassay,eds., Debtor et al, Plenum Press: New York, 3 (1985); Odell, inPrinciples of Competitive Protein-binding Assays; eds., Odell et al, J.Wiley & Sons, New York, 1 (1971); Quantitative Enzyme Immunoassay;Blackwell Scientifid, Oxford (1978); Engvall, in Enzyme Immunoassay,eds., E. Ishikawa et al, Igaku-Shoin, New York, 1 (1981); Jaklitsch, inEnzyme-Mediated Immunoassay, eds., Debtor et al, Plenum Press, New York,33 (1985)), and have been developed to detect a number of differentcompounds of environmental concern (see Immunochemical Methods forEnvironmental Analysis; ACS Symposium Series 442; Amer. Chem. Soc.,Washington, DC (1990); Mapes et al, Bull. Environ. Contam. Toxicol. 49,in press (1992); Immunoassays for Trace Chemical Analysis; ACS SymposiumSeries 451, Amer. Chem. Soc., Washington, DC (1990); Harrison et al, inBiotechnology for Crop Production, Hedin et al, eds., ACS SymposiumSeries 379; Amer. Chem. Soc.: Washington, DC, 316 (1988); Hammock et al,in Recent Advances in Pesticide Analytical Methodology; Harvey et: al,eds., ACS Symposium Series 136; Amer. Chem. Soc.: Washington, DC, 321(1980); Van Emon et al, in Analytical Methods for Pesticide and PlantGrowth Regulators Vol. XXII, 217 (1989); Albro et al, Tox. & Appl.Pharm. 50:137 (1979); Blewett et al, Bull. Environ. Contam. Toxicol.45:120 (1990); Bushway et al, Bull. Environ. Contam. Toxicol. 40:647(1988); Fleeker et al, in Immunoassays for Monitoring Human Exposure toToxic Chemicals; Vanderlaan et al, eds., ACS Symposium Series #451(1991); Gee et al, J. Agric. Food Chem. 36:863 (1988); Goh et al, Bull.Environ. Contam. Toxicol. 46:30 (1991); Harrison et al, Agric. FoodChem. 37:958 (1989); Jung et al, J. Agric. Food Chem. 37:1183 (1989);Jung et al, Pesticide Science 26:303 (1989); Thurman et al, Anal. Chem.62:2043 (1990)).

One of the most serious problems in environmental contamination is thepresence of polychlorinated biphenyls (PCBs). PCBs, as commerciallyavailable, exist as mixtures of PCB congenors containing variousmixtures of 209 different isomeric forms. These mixtures weredistributed commercially under the commercial name AROCLOR. A numberassigned to the AROCLOR designation indicates average percentchlorination of the PCB congenors in the product. Thus, AROCLOR 1260contains PCBs with an average chlorination of 60%, AROCLOR 1254 has anaverage chlorination of 54%, AROCLOR 1248 has an average chlorination of48%, AROCLOR 1242 has an average chlorination of 42%, and AROCLOR 1232has an average chlorination of 32%. The only one of the important toxicAROCLORs which does not follow the above rule is AROCLOR 1016, which hasan average chlorination of 41%. Toxicological data has indicated thatthe highly chlorinated PCBs are the most toxic to human health. The mostfrequently encountered toxic AROCLORs are AROCLOR 1260, AROCLOR 1254,AROCLOR 1016, AROCLOR 1232, AROCLOR 1242 and AROCLOR 1248. However,since the composition of PCB products varies from individual product toindividual product, from manufacturer to manufacturer within the sameproduct, and even from lot to lot within the same product, immunoassaytest development is difficult.

Mattingly et al, U.S. Pat. No. 5,145,790, disclose an immunoassay basedmethod for detecting polychlorinated biphenyls. However, the Mattinglyet al method requires the use of a combination of antibodies in order todetect both the highly chlorinated biphenyls, such as AROCLORs 1260 and1254, as well as the lower chlorinated biphenyls, including AROCLORs1016, 1221, 1232, 1242, and 1248. Thus, in order to use Mattingly et alin a screening program at a contamination site, one would be required toeither test each sample multiple times with different antibodies, or touse mixtures of antibodies in a test.

Under the Toxic Substances Control Act, the U.S. EnvironmentalProtection Agency (EPA) requires the cleanup of all spills anddischarges where the spilled material contains more than 50 ppm of PCBS.Contaminated surfaces are required to be cleaned to 10 μg/100 cm² or 100μg/100 cm², depending on the nature of the surface. To determine (1) ifa cleanup is necessary and (2) whether such a cleanup has beeneffective, the EPA requires that standard wipe tests be performed on thesurface. In 40 CFR 761.123 the EPA defines a "standard wipe test":

. . "Standard wipe test" means, for spills of high concentration PCBs onsolid surfaces, a cleanup to numerical surface standards and sampling bya standard wipe test to verify that the numerical standards have beenmet. This definition constitutes the minimum requirements for anappropriate wipe testing protocol. A standard size template (10centimeters (cm)×10 cm) will be used to delineate the area of cleanup;the wiping medium will be a gauze pad or glass wool of known size whichhas been saturated with hexane. It is very important that the wipe beperformed very quickly after the hexane is exposed to air. EPA stronglyrecommends that the gauze (or glass wool) be prepared with hexane in thelaboratory and that the wiping medium be stored in sealed glass vialsuntil it is used for the wipe test. Further, EPA requires the collectionand testing of field blanks and replicates.

In 40 CFR 761.130(e) the EPA also recommends a study by the MidwestResearch Institute (MRI) which describes a standard wipe test in moredetail. The report proposal leaves a considerable amount of latitude tothe analyst as to how the sampling and analysis are to be performed("Verification of PCB Spill Cleanup by Sampling and Analysis", pp.41-42).

Thus, an immunoassay method is needed which will provide reliable,accurate and fast results in the field for a wide range of PCBcontaminants in a single test using a single antibody, regardless ofmanufacturer, exact composition or matrix. Such an assay would increasethe efficiency of environmental site management activities such ascharacterization (mapping), remediation monitoring, and regulatorycompliance.

In addition, a standard PCB wipe test is needed which will providereliable, accurate and fast field results in accordance with EPAguidelines.

SUMMARY OF THE INVENTION

Accordingly, one object of the present invention is to provide a PCBimmunoassay method which is field compatible, fast and accurate.

A further object of the present invention is to provide a PCBimmunoassay method which gives minimal false negative results.

Another object of the present invention is to provide a PCB immunoassaymethod which detects PCB contamination derived from a wide variety ofunknown sources approximately equivalently, where such method isindependent of the detection of a single congenor in the mixture of PCBcongenors, since concentrations of single congenors vary betweencommercial PCB products.

Another object of the present invention is to provide a PCB immunoassaywhich is simple to perform and easy to interpret in a field orlaboratory setting by operators inexperienced in the art.

Another object of the present invention is to provide an anti-PCBmonoclonal (MAb) antibody which is useful in the above-mentioned PCBimmunoassay.

Another object of the present invention is to provide an anti-PCBmonoclonal antibody with specific reactivity towards PCB mixtures ofaverage chlorination of 32% or higher.

Another object of the present invention is to provide an anti-PCBmonoclonal antibody with specific reactivity towards AROCLOR 1260,AROCLOR 1254 and at least one member selected from the group consistingof AROCLOR 1016, AROCLOR 1232, AROCLOR 1242 and AROCLOR 1248.

Another object of the present invention is to provide a reagent (e.g.,an enzyme conjugate reagent) for use with the monoclonal antibody incompetitive binding type immunoassays.

Another object of the present invention is to provide immunogens whichelicit an anti-PCB monoclonal antibody response.

Another object of the present invention is to provide a kit forperforming a PCB immunoassay.

Another object of the present invention is to provide a method and kitfor performing a PCB immunoassay on a variety of sample matrices,including soil, oil, paint, condensate, water and solid surfaces.

Another object of the present invention is to provide a wipe test andwipe test kit useful in detecting PCB contamination on solid surfaces inaccordance with government regulations.

These and other objects of the present invention, which will becomeapparent from a reading of the description of the invention givenhereinbelow, have been found by the inventors to be satisfied by theirdiscovery of a monoclonal antibody which recognizes a molecularcharacteristic common to multiple components of a variety of PCBproducts, and to the inventors' discovery of immunogens which can beused to generate such a monoclonal antibody.

The various PCB products, such as the commercially available AROCLORs,differ from each other with regard to their particular chemicalcomposition. The chemical composition of the various AROCLORs has beenpublished by Capel et al, Chemosphere 14(5), 439-450 (1985) (forAROCLORs 1242, 1254 and 1260). However, the most toxic have in commonspecific structural elements, although the relative amounts of eachindividual congenor having the structural element or elements may varyfrom one PCB product to another. The antibody of the present inventionmolecularly recognizes the characteristic structural elements common tochemically diverse PCB products with relative equivalency, and cantherefore be used to detect the presence of PCBs (contamination) in asample since the concentration of the pool of characteristic compoundsdetected by the antibody is relatively constant in chemically disparatePCB products.

The present invention accordingly provides, among its variedembodiments, both an antibody and a test kit which incorporates theantibody. The kit is useful to detect the presence of PCBs(contamination) in a sample because the antibody exhibits approximatelyequivalent detection of PCB contamination, regardless of product,manufacturer, or diversity of chemical composition, and thereby providesan easy-to-use and highly reliable kit for environmental testingapplications.

BRIEF DESCRIPTION OF THE FIGURES

A more complete appreciation of the present invention and many of theattendant advantages thereof will be readily obtained as the samebecomes better understood by reference to the following detaileddescription when considered in connection with the accompanying figures,wherein:

FIG. 1 is a graphical representation of tests performed to measure soilmatrix effects on the immunoassay of the present invention between EnSys(a North Carolina clay soil), Sandy Loam and a methanol sample spikedwith PCBs.

FIG. 2 is a graphical representation of tests performed to measurevariability between different operators in performing the immunoassay ofthe present invention.

Both Figures demonstrate the sensitivity and specificity of the assay ofthe present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In one embodiment, the present invention provides an immunoassay fordetermining the presence of PCBs (contamination) in a sample (suspectedto contain PCBs). The immunoassay comprises:

(i) contacting a monoclonal antibody with specific reactivity towardsAROCLOR 1260, AROCLOR 1254 and at least one member selected from thegroup consisting of AROCLOR 1016, AROCLOR 1232, AROCLOR 1242 and AROCLOR1248, with a mixture of the sample and a reporter molecule reagent whichis cross reactive with the monoclonal antibody, to form an assaymixture;

(ii) incubating the assay mixture to allow competitive binding to theantibody between the PCBs, if present, in the sample and the reagent toform a monoclonal antibody complex; and

(iii) detecting the extent of binding of the reporter molecule reagentto the monoclonal antibody and correlating the amount of bound reportermolecule reagent to the amount of PCBs in the sample.

In another embodiment, the invention provides an immunoassay kit whichcomprises three basic components: (1) a monoclonal antibody withspecific reactivity towards AROCLOR 1260, AROCLOR 1254 and at least onemember selected from the group consisting of AROCLOR 1016, AROCLOR 1232,AROCLOR 1242 and AROCLOR 1248, (2) a reporter molecule reagent crossreactive with the monoclonal antibody and susceptible to detection, andoptionally (3) a signal-generating reagent.

To induce, in a vertebrate host, the formation of the present monoclonalantibodies that can be used to detect the characteristic structuralelement present in the most toxic PCBs, an immunogen is used whichcontains a derivative moiety which mimics the structural features of thespecific structural element present in these PCBs. The immunogen issynthesized, by standard methods, by coupling a derivative moiety to animmunologic carrier molecule.

Examples of suitable methods include coupling carbohydrates by periodateoxidation and coupling to amines by mixed anhydride chemistry (see Huruet al in Methods in Enzymology (Colowick and Kaplan, Eds.) 70:104-142,Academic Press, N.Y. (1980); Wilson et al in Immunofluorescence andRelated Staining Techniques (Knapp et al, Eds.) pp. 215-224,Elsevier/North Holland, Amsterdam (1978); and Nakane et al, J.Histochem. Cytochem. 22:1084 (1974) for periodate methods; and seeErlanger et al., J. Biol. Chem. 228:713 (1957); Greenstein et al inChemistry of the Amino Acids, Vol. 2, Wiley, N.Y. (1961); and Vaughan etal, J. Am. Chem. Soc. 74:676 (1952) for mixed anhydride methods.

Known immunological carrier moieties can be used, including albumin(e.g., bovine serum albumin), thyroglobulin (e.g., bovinethyroglobulin), hemocyanin (e.g., keyhole limpet hemocyanin), polyaminoacids and other molecules having a minimum size, complexity, andforeignness to the host animal. Carriers are usually of the class ofproteins, polypeptides or peptides having a molecular weight of at least1,000 daltons and preferably >10,000 daltons. Carrier molecules may havea reactive group(s) available for covalent conjugation of thederivative. R-groups (e.g., COOH, NH₂) of amino acids or sugar moietiesof glycoprotein are often used for this purpose in the synthesis ofimmunogens.

Preferably, the immunogen is a compound of a derivative moiety of theformula ##STR1## wherein X and Y independently represent a halogen n isan integer from 0 to 5,

m is an integer from 0 to 4, wherein n and m cannot both be 0;

R₁ is ##STR2## or a single bond R₂ is ##STR3## wherein R₃ and R₄ areeach independently hydrogen, C₁ -C₂ alkyls, linear, branched, or cyclicC₃ -C₆ alkyls; and

p is O or an integer from 1 to 4;

linked to an immunogen carrier molecule.

Preferably X and Y are chloro with n being from 2 to 5 and m being from2 to 4. A most preferred derivative moiety is ##STR4## wherein X and Yare chloro, n is from 2 to 5, m is from 2 to 4 and p is 0 or an integerfrom 1 to 4.

Small environmental chemicals, such as the PCB congenors, by themselvesare too small to stimulate an immune response and elicit an antibodyresponse. To elicit an antibody response, derivatives of smallmolecules, such as a PCB congenor, are attached through a chemicalbridge to a much larger "carrier" molecule prior to immunization.According to the invention, the small molecules and accompanying bridgestructures of the derivative are designed so that they mimicimmunologically at least one common and prevalent congenor present inthe PCB molecular pool of the most toxic AROCLORs. A number ofmodifications to the bridge structure (R₁ --(CH₂)_(p) --R₂) are possibleand can produce similar results. Any vertebrate is a suitable host forthe immunization procedure, such as mice, rats, dogs, pigs and otherdomestic animals, with mice being preferred. Additionally, in vitroimmunization can be used, with animal cells, including human cells, asthe immunization host.

The immunoassay method of the present invention uses ahybridoma-derived, monoclonal antibody. The monoclonal antibody isselected using a multi-parameter screening process. A reactive panelprofiling system is developed to identify and select appropriate celllines and monoclonal antibodies on the basis of sensitivity, PCB productrecognition characteristics, and freedom of interference from confirmednegative soil samples or cross-reacting compounds. The candidatemonoclonal antibody reagents are characterized by their ability toselectively bind to a defined pool of molecules having the specificstructural element, which are present in targeted PCB products (e.g. thevarious AROCLORS). The selected antibody allows the method to rapidlydetect toxic PCB contamination, regardless of manufacturer orchlorination level, at, or above, the regulatory level (i.e., aconcentration of 1 ppm in soil at a >95% confidence level), with lowerconcentrations detectable at lower confidence levels. The antibodyoperates through the selective recognition of a specific molecularstructure element found in a plurality of compounds present in all PCBcompositions.

The reagent of the present invention, preferably containing a hapten anda reporter moiety, which in a referred embodiment is an enzyme conjugatereagent, is cross reactive with the monoclonal antibody and capable ofproviding a detectable change in the sample solution being assayed,either alone or in combination with a co-reagent. Preferably, thehapten-reporter reagent comprises a compound of formula ##STR5## whereinX and Y independently represent a halogen n is an integer from 0 to 5,

m is an integer from 0 to 4, wherein n and m cannot both be 0;

R₅ is ##STR6## or a single bond R₆ is ##STR7## wherein R₇ is hydrogen,C₁ -C₂ alkyls, linear, branched, or cyclic C₃ -C₆ alkyls; and

p is O or an integer from 1 to 4;

linked to a reporter molecule.

A preferred enzyme (reporter moiety) for use in the enzyme-conjugatereagent of the present invention includes an enzyme selected from thegroup consisting of horseradish peroxidase, alkaline phosphatase,β-galactosidase, glucose oxidase, G6-PDH (glucose-6-phosphatedehydrogenase) and urease, with horseradish peroxidiase being mostpreferred.

A more preferred enzyme conjugate reagent is a compound of formula##STR8## coupled to horseradish peroxidase (HRP).

The enzyme conjugate is prepared using conventional methods.Illustratively, numerous functional groups on enzyme molecules (e.g.amino, sulfhydryl, carboxyl, carboxamide, tyrosyl, sugars) offerconvenient points for the attachment for the ligands. As is known,enzymes enhance the sensitivity of the method by the catalyticamplification of the detection signal. A single molecule of the enzymescommonly used in immunoassay methods will convert approximately 106molecules of a substrate into a product within one minute at ambienttemperatures. Catalysis is a function of the conformation at theenzyme's catalytic site, and it is this conformation, and the alignmentof certain amino acid residues at spatially significant positions, thatinfluences its rate and selectivity. The catalytic site is maintained bynon-covalent (i.e. hydrophobic, hydrogen bonding, ionic and Van derWaals interactions) and covalent (i.e. disulfide) forces, and can beinfluenced by temperature, the binding of ions, chaotropic agents,detergents, lipids, etc.. It is therefore important to normalize andcorrect for anticipated variations in the reaction environment.

The enzyme conjugate of the present invention must be cross-reactivewith the anti-PCB antibody of the present invention and capable ofinducing a detectable change in the sample by reaction with a signalproducing substrate or reagent used in the immunoassay.

The immunoassay-based system of the present invention uses a signalproducing reagent which, upon reaction with the bound enzymeconjugate/antibody complex after incubation, provides a detectablesignal indicating the presence or absence of PCB contamination in thesample being tested. Suitable signal producing reagents include ahydrogen peroxide/tetramethyl benzidine pair, or a hydrogenperoxide/phenylene diamine pair, in combination with an enzyme conjugatereagent containing horseradish peroxidase, o-nitro-β-D-galactopyranoside(ONPG) in combination with an enzyme conjugate reagent containingβ-galactosidase, o-nitrophenylphosphate in combination with an enzymeconjugate reagent containing alkaline phosphatase and glucose incombination with glucose oxidase or G6-PDH. The method of the presentinvention preferably uses the combination of a hydrogen peroxide (H₂ O₂)substrate solution with a tetramethylbenzidine (TMB) chromogen, with thecombination producing a blue chromophore upon oxidation of TMB by H₂ O₂.

An important characteristic of the immunoassay of the present invention,as well as with any field screening method, is the minimization of falsenegative results without producing excessive false positive data.Statistically, therefore, the standard concentration should not be setat the concentration of interest, but below it. Setting the standardconcentration to coincide with the desired detection concentration wouldresult in a 50% false negative incidence at that desired concentration.To minimize this effect, standards used in the immunoassay of thepresent invention should be offset below the target concentration toproduce a >95% confidence level of detecting contamination at, or above,the desired level. For example, to obtain a method which gives resultshaving a >95% confidence level for detection of PCBs at 5 ppm, a 2.5 ppmstandard could be used. The standard is set below the target level. Theactual concentration of standard used is a function of the dose-responsecharacteristics of the assay and the overall interference and precisionof the method.

Immunoassay methods have conventionally been used to test liquidmatrixes such as blood, urine, and water. This method is designed totest also liquid oil-based matrices, solid matrices and solid surfaces.However, the testing of liquid oil-based matrices and solid matricesrequires that the issues of sample collection, dispersion, extractionand clarification be addressed and integrated with the immunoassaycomponent.

While the assay of the present invention may be performed on samples ofa wide range of qualities, a reproducible, particulate-free, extract ispreferred for optimum results. The extraction and recovery of a compoundfrom soil requires the selection of an appropriate solvent system,adequate sample dispersion, sufficient time for partitioning,non-invasive clarification and compatibility with the subsequentimmunochemistry. However, current sample processing by analyticalmethods utilizes solvents that are incompatible with immunochemicalmethods and is slow and expensive.

One suitable method for soil sample preparation involves the collectionof a 10 g sample measured with a small battery-operated balance. Thesample is then transferred into a dispersion vial containing a suitableorganic solvent, such as methanol, and conventional dispersing pellets,and is subjected to a one minute manual agitation for adequatedispersion and partitioning of the analyte into the solvent. Filtrationof the sample suspension to produce a particulate-free extract can beaccomplished using a fingertip-operated filter unit fitted withnon-adsorbing filters. The clarified extract is then ready for testingwith the immunoassay of the present invention following dilution orother removal of the solvent. Solvent-resistant antibody reagents couldbe used to eliminate or limit the need for the solvent or dilution step.

Buffers, detergents (e.g. Tweens, Tritons, etc.) or solvents, usedalone, or in combination, have proven to be effective for extractionfrom soil samples. Analytical methods for the analysis of solid wasterely upon gravimetrically collected samples, and results are reported ingravimetric units. Volumetric sampling for solid waste should preferablybe avoided because of the potential bias (up to 200%) that may be causedby the specific gravity of the sample.

The present immunoassay method permits the simultaneous testing of soilsamples for contamination at multiple concentrations to facilitate theconstruction of contamination contour profiles. The method is preferablyfor screening purposes and several safeguards have been incorporated tominimize the incidence of false negative results.

Common oil-based matrices for PCBs include transformer oils such asmineral oils and non-PCB dielectrics, motor oils, silicone oils, fueloils, organic solvent waste streams of synthetic processes, chlorinatedsolvents from the dry cleaning and electronics industries, gas pipelinecondensates and phthalate ester based oils, as well as aqueous basedpaints and condensates that contain oil-based contaminants. The EPArequires used oils which are destined for recycling to contain less than2 ppm PCBs, while waste oils or solvents destined for incineration mustcontain less than 50 ppm PCBs. Unfortunately, most of the aboveoil-based matrices contain oils or solvents which are incompatible withaqueous immunochemistry methods. Accordingly, one embodiment of thepresent invention provides a sample processing component for use inpreparing samples from oil-based matrices, such as those listed above,for use in the PCB immunoassay of the present invention.

Due to the lipophilic nature of PCBs, it is extremely difficult toremove the PCBs from oil-based matrices by mere extraction. In order toremove the PCBs from the oil-based matrices, a solvent must be usedwhich: (1) is immiscible with alkanes, especially higher molecularweight alkanes and (2) provides efficient partitioning of PCBs from thematrix. Since extraction alone has proven unacceptable, the extractionsolvent must also be compatible with the following processingoperations: (1) resistant to attack from strong acids, oxidants, alkaliand reducing agents, (2) have utility in chromatographic methods,especially normal phase thin-layer chromatography (TLC) on silica platesand (3) have sufficient volatility or susceptibility to analytereconcentration methods. A suitable solvent which meets all of the aboverequirements is acetonitrile.

In order to remove PCBs from oil-based matrices in a manner whichprovides a workable immunoassay using the method of the presentinvention, a multistep sample processing protocol is used. Theprocessing protocol comprises:

(1) Extraction of the oil-based sample with the extraction solvent,preferably acetonitrile, in a volume ratio of solvent to samplesufficient to balance sensitivity desired (provided by small extractionsolvent volumes) with matrix variability (compensated by largeextraction solvent volumes). The preferred volume ratio of solvent tosample is approximately 9:1.

(2) Oxidation of a portion of the extract obtained, by treatment with anoxidizing agent and an acid. Suitable oxidizing agents includechromates, permanganates, periodates, persulfates, cerium (IV) salts,cobalt (III) salts and manganese (III) salts, with ceric ammoniumnitrate being preferred. Suitable acids include the strong mineral acidswith sulfuric, perchloric, nitric and tetrafluoroboric acids beingpreferred, with 0.5 to 1.0 ml, preferably 1.0 ml, of 70% HClO₄ used per1 ml of extract in a most preferred embodiment.

(3) Quenching and concentrating the oxidized extract by combining theoxidized extract with about 4 volumes of an aqueous salt solution,wherein the aqueous salt solution is preferably a solution of sodiumchloride, sodium sulfate or sodium nitrate at a concentration of from 2Mto 5.5M, followed by extraction into 0.1 volumes of a suitable solventwhich is immiscible with the salt solution, such as linear, branched orcyclic hydrocarbon solvents having from 5 to 8 carbons or methylenechloride, with isooctane being most preferred. This extraction procedureis a modification of the procedure of Jenkins et al Anal. Chem. 63, 1341(1991).

(4) Chromatographing the resulting isooctane solution using normal phasechromatography, preferably normal phase TLC, for example, on achromatographic plate having as a chromatographic medium a memberselected from silica, fluorosil, alumina and bonded phase silica, withsilica being preferred, using a suitable developing solvent, whichpreferably gives retention factors (R_(f) s) for the various AROCLORs offrom 0.32 to 0.68. A preferred elution solvent is a linear, branched orcyclic volatile hydrocarbon solvent having from 5 to 8 carbons, withisooctane being most preferred. In a preferred embodiment the TLC plateis preeluted with methanol to remove possible interfering substancesfrom the plate, prior to elution of the sample. After development of thechromatographic medium (e.g., the TLC plate), the plate is extractedwith methanol to remove the PCBs and the methanol extract is subjectedto the immunoassay of the present invention.

When testing samples containing these types of matrices without theabove processing protocol, the presence of turbidity in the first stepof the immunoassay is an indicator for possible false negative results.Thus, if the buffer solution of the first step becomes turbid onaddition of the sample, a negative result should be viewed with caution.

The basic characteristics of specificity and equivalent detection of theassay of the present invention are a result of the monoclonal antibody(MAb) and the reporter molecule reagent. The assay providesapproximately equal recognition of a variety of PCB products (e.g., thevarious AROCLORs), approximately equal recognition of PCBs fromdifferent manufacturers having different chemical compositions and withdifferent chlorination levels, low cross-reactivity with unrelatedcompounds and is not significantly affected by normally foundconstituents of negative soil extracts.

Conventional hybridoma techniques are employed to prepare the anti-PCBmonoclonal antibody (MAb) for the assay of the present invention, by theuse of the immunogen reagent of the present invention. The method forproducing MAbs is extremely powerful and allows for the preparation of adefined and reproducible Ab reagent with a unique, chosen specificity(see Milstein, Cancer, 49:1953-1957, (1982)). The production ofpolyclonal antisera is much less controlled since polyclonal antiseraare the product of numerous Ab populations having varying specificityand sensitivity characteristics. An immunoassay for PCB products hasbeen developed using polyclonal antisera, but has been shown to exhibita number of problems, such as variability in specificity and recognitionrequiring very lengthy sequential protocols. MAbs are also homogeneouswith a defined specificity, unlike polyclonal antisera which contain amixed population of antibodies. Hybridoma cell lines are immortal whenstored under appropriate conditions and provide access to an unlimitedsupply of MAb reagent that provides performance characteristics to thetest system that will be exactly the same from lot to lot.

A unique strategy is used for antibody production. This strategyinvolves developing an antibody to one or more specific structuralelements that are present on a defined set of compounds in PCB products.Because of the chemical complexity of PCB products and the tendency ofthe composition to vary between products and among manufacturers, theantibody is designed to recognize defined structural elements present incompounds contained in each of the various AROCLOR compositions.Recognition of a panel of compounds, containing the defined structuralelements instead of a single compound, allows the assay of the presentinvention to average out the concentration difference of any singlecomponent present in the different types of PCB products. Additionally,recognition of a collection of compounds rather than a singleconstituent contributes significantly to the sensitivity of the assay.

Preferably the pool of compounds selected for detection by the assay arepredominantly a PCB congenor chlorinated with 4, 5 or 6 chloro groups.

The immunogen of the present invention is used to immunize suitablehosts, such as mice.

Hosts responding to the immunization protocol are selected as splenocyte(or lymphoid cell) donors for hybridoma production. A culture of immunesplenocytes in a defined tissue culture medium, such as HAT, withconventional modified myeloma cells, capable of providing a hybridoma,provides the fusion products s--s, s-m, and m--m (with s=splenocyte andm=myeloma cell). Within tissue culture medium the s--s fusion productnormally has a short lifetime and dies within days. Also, the m--mfusion product has a very short lifetime in the tissue culture mediumused, lacking the metabolic components needed for DNA synthesis. Howeverthe s-m fusion product (or hybridoma) survives in tissue culture andretains the Ab-producing characteristics of the s,plenocyte parent, andthe high rate of growth and relative immortality of the myeloma cellparent. This hybridoma cell line replicates itself readily in cultureproducing daughter cells that provide a reproducible, homogeneous, andconsistent supply of the monoclonal anti-PCB antibody of the presentinvention.

The structure of the reporter molecule reagent can have a significantinfluence on immunoassay performance. Both the derivatized ligand andthe bridge play an important role in antibody binding. Antibody andreporter molecule reagent pairs should be able to satisfy the followingcriteria:

Recognition and displacement and prerequisite sensitivity for AROCLORsand other PCB products in a sample

Low cross-reactivity to non-related compounds

Approximately equal sensitivity for different PCB products, i.e., toxicAROCLORs

Insignificant negative soil matrix effects Antibody and reportermolecule reagent pairs which best satisfy the above criteria are mostsuitable for the assay of the present invention.

The immunoassay of the present invention is performed according to thefollowing procedure. Monoclonal anti-PCB antibody is immobilized in anassay vessel, or other solid support, preferably to the bottom of apolystyrene tube, at a concentration, of from 0.005 to 20 μg/tube,preferably 0.1 to 0.4 μg/tube. The concentration and affinity of theantibody for the sample molecules and reporter reagent moleculesdirectly influences the overall sensitivity of the final method. High,equivalent, affinity, and minimal non-specific signal generation,produces assays having superior sensitivity, with affinity bindingconstants, Ka, from 10⁴ -10¹² L/mol, preferably 10⁴ 10⁹ L/mol.

The assay of the present invention compares a sample to be tested with astandard solution containing a compound cross-reactive with the antibodyof the present invention, which is present in the standard solution inan amount sufficient to provide a 95% confidence level for detection atthe desired level.

By way of example, the following describes a simultaneous test of anegative sample, a sample containing ≧5 ppm of PCBs, and a standardsolution containing the equivalent of approximately 2.5 ppm of AROCLOR1248. Initially, following extraction from soil, the sample extract andstandard are added to separate, and identical, antibody-coated testtubes. To each tube is added an equal volume of the reporter moleculereagent solution. The tubes are then allowed to incubate at ambientconditions for approximately ten minutes.

During the incubation period sample molecules and reporter reagentmolecules compete for the limited number of antibody binding sites thatare available on the bottom of each of the tubes. The antibodyconcentration present is insufficient to permit the binding of all ofthe sample and reporter reagent molecules simultaneously, and asituation somewhat analogous to the game of musical chairs exists, withthe limited antibody binding capacity serving as the chairs in thisexample. The concentration of reporter molecule reagent immobilized ineach tube after incubation is inversely proportional to theconcentration of PCBs in the sample or standard. The standard in theStandard tube limits the binding of reporter molecule reagent, theNegative sample permits more reporter molecule reagent to bind (relativeto the standard), and the positive sample limits the binding of thereporter molecule reagent (relative to the standard). At the end of the10 minute incubation period, the tubes are washed leaving only thereporter molecule reagent that was retained by the immobilized antibodyon the bottom of each tube.

The bound reporter molecule reagent/antibody complex remaining is nextused to produce a detectable signal. Upon addition of a signal formingreagent, preferably a substrate/chromogen reagent, the enzyme moleculescatalyze the formation of a detectable signal. The signal that isgenerated is directly proportional to the concentration of reportermolecule reagent present. A preferred substrate/chromogen reagent is H₂O₂, and tetramethyl-benzidine (TMB) which, when used with a preferredreagent wherein the reporter molecule is the enzyme horseradishperoxidase, reacts with the enzyme portion of the reagent, facilitatingthe oxidation of TMB by H₂ O₂ and generating a blue color.

Thus, the negative sample tube rapidly produces a solution that isvisibly darker (i.e. greater absorbance) than the standard tube. Thepositive test sample produces a solution having less color (i.e. lowerabsorbance) than the standard tube. By comparing the signal of thesample tubes to the signal of the standard tube using a battery-operatedcomparative photometer, optionally included with the kit of the presentinvention, samples containing greater than or equal to the desired levelof contamination can be detected with >95% confidence. In thiscompetitive ELISA method the final absorbance is inversely proportionalto the ligand concentration present in the test sample.

The assay method of the present invention can process multiple samplesin less than 30 minutes. The method is self-contained, field-compatibleand does not require refrigeration or use hazardous components. Thedetection level can be set at the users discretion, with 0.3 ppm as themaximum sensitivity obtainable with the current configuration using asolid matrix sample and 15 ppb for aqueous samples.

The kit of the present invention may be configured for analysis of soilsamples, oil matrix samples, aqueous samples, and solid surfaces. Thekit of the present invention contains five basic components: (1) asample processing component for preparing the sample for testing by theassay method (described above for soil samples), (2) at least one assayvessel, preferably a plurality, containing an amount of an immobilizedanti-PCB monoclonal antibody, equivalent amounts when a plurality ofvessels is used, (3) a standard solution containing a specific level ofPCBs in a suitable solvent, (4) a reporter molecule reagent which isreconstituted to form a solution containing a reporter reagent which iscross-reactive with the monoclonal anti-PCB antibody, in a suitablesolvent, where the reporter reagent is preferably a lyophilizedenzyme-conjugate reagent, and (3) means for forming and detecting asignal indicating the presence or absence of PCB based contamination inthe sample being tested. Optionally, the kit contains one or more of thefollowing additional components: (6) wash solutions for performing thewash step of the assay method, (7) weighing boats into which the sampleis weighed using (8) a battery-operated scale, (9) one or more filtersfor preparing a clarified extract sample, (10) one or more dilutionvials, (11) substrate solution and (12) stop solution to halt signalformation by the signal-forming reagent. Preferably the assay isprovided in a coated-tube format in which the means for forming anddetecting a signal comprises the use of a substrate/chromogen reagentwhich forms a detectable colored signal with the level of PCBcontamination determined by comparison of the color developed by thetest samples to the color of the kit standard. In order to eliminatetemperature variations, timing errors and operator mistakes, thestandard is preferably run in parallel with the samples. Therefore, anyassay variations will equally affect both the standard and the samples.Using this format with the standard run in parallel, the assay has thefollowing characteristics:

1. The sensitivity as currently configured for soil samples is 0.35 ppmfor AROCLOR 1260, 0.33 ppm for AROCLOR 1254, 1.00 ppm for AROCLOR 1248,1.57 ppm for AROCLOR 1242, 3.25 ppm for AROCLOR 1232 and 3.50 ppm forAROCLOR 1016.

2. Different soil matrices cause little interference in the assay.

3. The test is reproducible from lot-to-lot, day-to-day, andperson-to-person.

4. The assay is operable over a temperature range of 4°-30° C.,preferably 15°-30° C.

5. Storage at room temperature (up to 30° C.) is acceptable.

The immunoassay of the present invention has many advantages, includingfield screening compatibility, speed in obtaining a result, and a lackof false negative results. Specific characteristics had to be integratedinto the method in order to achieve these advantages. The first was tominimize the incidence of false negative results. A false negative testdoes not detect a contaminated sample and therefore, can be potentiallydangerous both to the customer and the environment. A false positive, onthe other hand, might be inconvenient but not dangerous. Therefore, theimmunoassay of the present invention was designed to detect positivesolid matrix samples containing ≧1 ppm of PCBs with a minimum of falsenegative results. This is accomplished by using a standard containingthe equivalent of AROCLOR 1248 in soil at 0.7 ppm to detect 1 ppm PCBwith a 95% confidence level. Preferably, two standards are testedsimultaneously with each assay, both to minimize false negative resultsand to provide an internal quality control parameter to control for theintegrity of the chemistry and performance of the operator.

The immunoassay has also been simplified by making it a semiquantitativetest using a standard at a single concentration. The use of one standardconcentration greatly simplifies the test protocol and eliminates thedata manipulation and expensive instrumentation that are normallynecessary for quantitative tests. The test becomes a semiquantitative"yes/no" test that determines whether a sample contains greater or lessthan a set level of PCB contamination, depending on the concentration ofthe assay standard. The use of multiple standards at varyingconcentrations allows one to perform a quantitative assay by comparisonto the signals produced by the samples on a concentration vs. signalplot.

In the wipe test of the present invention, the sample processingcomponent (1) is an extraction vessel containing an extraction solventand a wiping means. In addition to the extraction vessel and itscontents, the wipe test also includes a wipe template which provides acontrolled surface area to be wiped by the wiping means.

The extraction vessel may be made from any suitable material which isnonreactive with the extraction solvent contained therein. Suitableextraction solvents include lower alcohols, such as methanol, ethanol,and isopropanol with methanol being preferred. The extraction solventused must not leach interfering substances from the wiping means. Thewiping means may be a woven or nonwoven cloth material made fromsynthetic or natural fibers, which are inert in the presence of theextraction solvent and PCBs.

Cotton gauze or glass wool are preferred, with the preferred size being4 in by 4 in. A suitable wiping means is made from DURX 670, soldcommercially by Berkshire Manufacturing Co.

The wipe template is used to provide a controlled surface area forwiping in order to correlate with the EPA requirements, noted above.While the wipe template may provide any surface area as the controlledarea, it is preferred that the template specifically define a 100 cm²area, preferably of dimensions 10 cm×10 cm. Use of other surface areatemplates would require correction of the results obtained in order tocorrelate to the EPA standard.

In performing the wipe test with the assay of the present invention, thewiping means is removed from the extraction vessel and excess solvent issqueezed from the wiping means back into the vessel. The wipe templateis held against the surface to be tested and the exposed surface iswiped using the wiping means. When the wiping means is a 4 in by 4 incloth, the surface is wiped using both sides of the wiping means. Thewiping means is then returned to the extraction vessel and the vesselrecapped and shaken to extract the PCBs from the wiper. A sample of theextract is then decanted and filtered before use in the immunoassay ofthe present invention. The wipe test of the present invention provides apositive result whenever a surface wipe recovers 10 μg of PCBs from 100cm² of surface, thus meeting or exceeding the EPA requirements. Indeveloping the PCB wipe test, it was desired to adapt the wipe procedureto the PCB immunoassay of the present invention without anymodifications to the immunoassay procedure. This is accomplished bycorrelating any matrix effects in the wipe and soil tests. No systematicdifferences between the wipe matrix and various soil matrices werefound.

The immunoassay of the present invention makes possible the rapidscreening of numerous samples in the field at the site. The rapid,on-site screening of multiple samples is a significant advantagecompared to the standard analytical GC or IR methods. The quickscreening of samples allows quick answers to important questionsconcerning the project, such as, the area of contamination, has the areabeen remediated to acceptable levels, and does additional contaminatedmaterial need to be removed? With the immunoassay method of the presentinvention, personnel, time and equipment can be used morecost-effectively.

Having generally described this invention, a further understanding canbe obtained by reference to certain specific examples which are providedherein for purposes of illustration only and are not intended to belimiting unless otherwise specified.

EXAMPLE Example 1 Preparation of Assay Components Preparation ofMonoclonal Antibody

Female Balb/c mice received a primary subcutaneous immunization with 50ug of PCB derivatized to bovine serum albumin in adjuvant (MPL+T DMEmulsion, RIBI Immunochem Research, Inc., Hamilton, Mont.). Subcutaneoussecondary injections (25 ug) were given at day 21, 35, and then monthly.The production of monoclonal antibodies was performed according toconventional procedures (see Milstein, Cancer, 49:1953-1957 (1982)).Hybridoma cultures producing monoclonal antibodies recognizing freehapten by EIA were cloned by limiting dilution. Clone F40-11G6 wasselected based upon sensitivity, PCB product recognition characteristicsand freedom of interference from confirmed negative samples orcross-reacting compounds. This clone was deposited, in accordance withthe Budapest Treaty, with the American Type Culture Collection (ATCC) onOct. 28, 1997 as ATCC No. HB-12421.

Horseradish peroxidase conjugate reagent

Horseradish peroxidase (HRP) conjugate reagent was prepared with ahydrazide derivative of 2', 4'-dichlorobiphenyl-1-carboxylic acid usingthe conventional periodate method (see Nakane et al J. Histochem.Cytochem. 22:1084 (1971)).

Antibody Coated Tubes

Antibody coated tubes were prepared by passively coating polystyrene(12×75 mm, NUNC) tubes with the monoclonal F40-11G6 antibody diluted inPBS and incubating overnight.

Example 2 PCB Immunoassay Procedure--1 ppm in Soil

The immunoassay method of the present invention has been demonstrated asfollows:

10 g of a soil sample were placed in an extraction vial containing 20 mLof methanol and 5 steel pellets (6mm) and the vial and its contentsshaken for 1 minute to disperse the soil particles and extract the PCB.The sample was then filtered to clarify the sample using a filter ofglass fiber and 0.45μ Teflon. 45 uL of the clarified filtrate sample wasmixed with 955 μl of 10 mM PBS (pH=7.4) and the resulting mixture addedto lyophilized enzyme conjugate reagent (0.3 μg) and mixed. At the sametime, 2×45 uL of the AROCLOR 1248 standard are similarly treated. Thesample and the two standards were then poured into three identicalantibody coated tubes, respectively, and the resulting solution mixed.The resulting solution was incubated for 10 minutes at ambienttemperature and then washed 4× with a wash buffer (detergent-salinesolution). 250 uL of tetramethylbenzidene solution (KP Laboratories,Cat. No. 50-76-02) was added along with 250 uL of hydrogen peroxidesolution (KPL, Cat. No. 50-65-02) and the resultant solution mixed.After incubating for 2.5 minutes the color development reaction wasstopped with 250 uL of stop solution (1M sulfuric acid).

The optical density (OD) of the test sample was compared to the OD ofthe AROCLOR 1248 standard (0.7 ppm) which was tested simultaneously withthe sample. If the sample OD is less than the standard OD, the samplecontains greater than 1 ppm PCBs. If the sample OD is greater than thestandard OD, the sample contains less than 1 ppm PCBs. Otherconcentration ranges can be assessed by diluting the test samples withsuitable diluents, as long as they are non-reactive with the componentsof the assay.

The sensitivity of the soil test was established to be 1.0 ppm for PCBin soil with >95% confidence for AROCLOR 1248. To develop the test fordetection at this level, the concentration of AROCLOR 1248 in the kitstandard was set to coincide with the mid-point of the immunoassay doseresponse curve, the most precise portion of the curve. Also, theantibody concentration immobilized on the tubes and the sample dilutionprotocol were adjusted so that samples containing 1 ppm or more PCBswere positive (i.e. lower absorbance) versus the absorbance of the kitstandard.

Matrix interference in soils was assessed by determining the assaysensitivity (recovery of spiked samples) in soil extracts confirmed tobe negative by GC analysis. Since the sensitivity of the assay wasbarely changed when spiked negative extracts were compared to spikedmethanol samples (FIG. 1), it was concluded that there was negligiblesoil matrix interference.

Another criterion, in demonstrating the performance of the assay of thepresent invention, was to determine the sensitivity to othercross-reacting compounds commonly found at PCB sites and compoundshaving similar structures.

These compounds were tested for their reactivity with the present 1 ppmPCB immunoassay. Table 1 shows the results obtained, the concentrationproviding a positive result compared to the kit standard and thecalculated percent cross-reactivity. Overall, the compounds were eachless than 1% cross-reactive, with one exception at 2%, indicatinghigh-specificity for PCBs in the present method.

                  TABLE 1    ______________________________________    CROSS-REACTIVITY TABLE                    CONCENTRATION                    GIVING A                    POSITIVE RESULT;                                  PERCENT                    SOIL EQUIVALENT                                  CROSS-    COMPOUNDS       (PPM)         REACTIVITY    ______________________________________    BENZENE         >10,000       <0.01%    1-CHLORONAPHTHALENE                    >10,000       <0.01%    O,P-DDT         >10,000       <0.01%    9,10-DICHLORO-  >10,000       <0.01%    ANTHRACENE    DIESEL          >10,000       <0.01%    ENVIROTEMP      >10,000       <0.01%    GASOLINE        >10,000       <0.01%    HEXACHLOROBENZENE                    >10,000       <0.01%    METHYLENE CHLORIDE                    >10,000       <0.01%    NAPHTHALENE     >10,000       <0.01%    PENTACHLOROBENZENE                    >10,000       <0.01%    PENTACHLOROPHENOL                    >10,000       <0.01%    R-TEMP          >10,000       <0.01%    TRICHLOROETHYLENE                    >10,000       <0.01%    2,4,6-TRICHLOR-p-                    >10,000       <0.01%    TERPHENYL    XYLENES         >10,000       <0.01%    2,4-D           1,000-10,000  <0.1%    HALOWAX 1013    1,000-10,000  <0.1%    HALOWAX 1051     100-1,000    <1%    BIFENOX          100-1,000    <1%    2,4-DICHLOROPHENYL                     100-1,000    <1%    BENZENESULFONATE    SILVEX           100-1,000    <1%    1,2,4-TRICHLOROBENZENE                     100-1,000    <1%    2,4-DICHLORO-1-NAPTHOL                    50            2%    TETRADIFON      10-100        <10%    ______________________________________

AROCLOR Recognition

Dose response curves were generated using the various commercial AROCLORcompositions. Table 2 shows the results of 3 sets of duplicate doseresponse curves and the mean ED50 obtained for each sample.

                                      TABLE 2    __________________________________________________________________________    AROCLOR SENSITIVITY COMPARISON    PCB MONOCLONAL TEST              1260                  1254                     1248                         1242 1232                                  1016                                      1221    __________________________________________________________________________    DETERMINATION 1              34.20                  32.10                     95.20                         176.00                              324.00                                  352.00                                      4,174.00    2         32.00                  34.60                     104.50                         160.00                              377.00                                  346.00                                      4,013.00    3         36.40                  29.00                     92.90                         122.00                              251.00                                  325.00                                      5,354.00    MEAN ED50 (ppb)              34.20                  31.90                     97.50                         153.00                              317.00                                  341.00                                      4,514.00    STD DEV   2.20                  2.81                     6.14                         27.74                              63.26                                  14.18                                      732.19    SENSITIVITY              0.35                  0.33                     1.00                         1.57 3.25                                  3.50                                      46.28    __________________________________________________________________________

Example 5

Reproducibility from person-to-person, day-to-day, and lot-to-lot wasinvestigated. Using sensitivity profile data accumulated from threelots, comparisons of three trained operators were made (FIG. 2). Thisgraph illustrates insignificant changes in specificity and sensitivityat the negative and 1 ppm levels. Another measure of operatorvariability is the reproducibility of the standard pairs, specificallythe difference in OD units between the two standards (see Table 3)

                  TABLE 3    ______________________________________    OPERATOR VARIABILITY,    REPRODUCIBILITY OF STANDARD PAIRS                         MEAN            NUMBER OF    DIFFERENCE   STANDARD    OPERATOR            OBSERVATIONS BETWEEN STDS DEVIATION    ______________________________________    1       59           0.058        0.059    2       67           0.060        0.057    3       82           0.073        0.053    ______________________________________

These results indicate no difference in reproducibility for trainedoperators.

Untrained Operators

This study had two purposes: 1) to determine if variation exists thatcauses differences in the interpretation of the test and 2) if the testis "useable" for the untrained operator.

Five operators, unfamiliar with running the test, were recruited toassay four sensitivity profiles consisting of a pair of kit standards, 0ppm, 0.25 ppm and 1.0 ppm (see Table 4). The tests (n=20) resulted in nofalse positives at 0 or 0.25 ppm and 5% false negatives (1 false resultout of 20) at 1.0 ppm.

                  TABLE 4    ______________________________________    UNTRAINED OPERATORS; VARIABILITY    OPERATOR     1       2       3     4     5    ______________________________________    N =          4       4       4     4     4    MEAN DIFF OF STDS                 0.18    0.19    0.11  0.15  0.07    (Std dev)    (0.05)  (0.14)  (0.05)                                       (0.08)                                             (0.06)    Bstd/Bo      0.71    0.59    0.49  0.50  0.59    False pos    0       0       0     0     0    0 ppm    False pos    0       0       0     0     0    0.25 ppm    False neg    0       0       1     0     0    1.0 ppm    ______________________________________

Also, the operators ran five field samples at two target levels, 1.0 and10.0 ppm. The results of these samples correlated with the GC resultsand the results of the present test kit run by the trained operators.

The results of these experiments clearly indicate the useability of thetest and the lack of operator variation.

Lot to lot variability was assessed by comparing the sensitivityprofiles generated from three validation lots. All three lotsdemonstrated a consistent number of false results with the followingpercent negative results at 0.40 ppm: Lot 1-100%, Lot 2-94% and Lot3-100%, and at 1.0 ppm: Lot 1-1%, Lot 2-4.1% and Lot 3-6.8%. Theseexperiments indicate that the product is reproducible and not subject tointer-person, day or lot variations.

Sensitivity profiles on spiked or negative soil were used to evaluatethe effective temperature range. The accuracy, sensitivity andspecificity of the test remained constant over the range of 4°-30° C.

Example 6

The assay test kit of the present invention showed little loss inperformance when stored at room temperature (20°-30° C.) for 3 months.Regression analysis evaluating signal decay and performance suggestslong-term stability (>6 months) when stored at room temperature.

Therefore, the data suggest that the assay method of the presentinvention is an acceptable, and more conservative, field screeningmethod for PCB contamination.

The advantages of immunoassay technology can be attributed to theunderlying lock and key binding principle and its compatibility withaqueous matrixes. This method does not require the chromatographicseparation of sample components, nor does it require that compoundsabsorb visible, infrared or UV for detection. Interferences from othercompounds are considerably less of a problem because of theconformational nature of the antibody binding process. Sample processingtime is significantly reduced, and the direct testing of aqueoussamples, or water-compatible extracts of soil, can be performed. Thetechnology offers a unique, and conservative, approach to fieldscreening. The incidence of false negative data is exceptionally low.Aspects that tend to interfere with immunoassay methods of this typetend to cause an overestimation of contamination, or false positiveresult by minimizing the binding of enzyme conjugate to antibody orsuppressing signal generation by the enzyme.

This method offers significant versatility and performance advantages.It is a convenient and effective new tool that can enhance theefficiency of site management activities and the utilization of ournational laboratory system.

Obviously, numerous modifications and variations of the presentinvention are possible in light of the above teachings. It is thereforeto be understood that within the scope of the appended claims, theinvention may be practiced otherwise than as specifically describedherein.

What is claimed as new and desired to be secured by Letter Patent of theUnited States is:
 1. A monoclonal antibody useful for detecting thepresence of PCB's in a sample, having specific reactivity towardsAROCLORs 1260, 1254, 1248, 1242, 1232, 1016 and 1221 that issubstantially the same as the monoclonal antibody produced by clone ATCCNo. HB-12421.
 2. The monoclonal antibody of claim 1, obtained by(i)providing an immune response in a vertebrate host by immunization withan immunogen comprised of a derivative moiety of formula: ##STR9##wherein X and Y independently represent a halogen n is an integer from 0to 5, m is an integer from 0 to 4, wherein n and m cannot both be 0; R₁is ##STR10## or a single bond R₂ is ##STR11## wherein R₃ and R₄ are eachindependently hydrogen, C₁ -C₂ alkyls, linear, branched, or cyclic C₃-C₆ alkyls; and p is O or an integer from 1 to 4; linked to an immunogencarrier molecule; (ii) preparing a hybridoma from the lymphoid cells ofsaid host; (iii) selecting said hybridoma which produces said monoclonalantibody; and (iv) obtaining said monoclonal antibody.
 3. An immunoassaykit useful for detecting the presence of PCB's in a sample, comprising amonoclonal antibody having specific reactivity towards AROCLORs 1260,1254, 1248, 1242, 1232, 1016 and 1221 that is substantially the same asthe monoclonal antibody produced by clone ATCC No. HB-12421.
 4. Animmunoassay kit useful for detecting the presence of PCB's in a sample,said kit comprising(i) a reaction vessel means containing an immobilizedmonoclonal antibody having specific reactivity towards AROCLORs 1260,1254, 1248, 1242, 1232, 1016 and 1221 that is substantially the same asthe monoclonal antibody produced by clone ATCC No. HB-12421, (ii) astandard solution comprising at least one of AROCLOR 1260, AROCLOR 1254or AROCLOR 1248, and a solvent; (iii) a hapten-reportermoiety-containing reagent which is cross-reactive with said antibody,and a suitable solvent; and (iv) means for causing said reporter moietyto generate a signal and means for detecting the signal.
 5. The kit ofclaim 5, wherein said hapten-reporter moiety-containing reagent is acompound of formula ##STR12## wherein X and Y independently represent ahalogen n is an integer from 0 to 5,m is an integer from 0 to 4, whereinn and m cannot both be 0; R₅ is ##STR13## or a single bond R₆ is##STR14## wherein R₇ is hydrogen, C₁ -C₂ alkyls, linear, branched, orcyclic C₃ -C₆ alkyls; and p is O or an integer from 1 to 4; linked to areporter molecule.
 6. The kit of claim 4, wherein said standard solutioncomprises from 0.6 to 10 ppm of said compound.
 7. The kit of claim 5,wherein said reporter molecule is an enzyme selected from the groupconsisting of horseradish peroxidase, alkaline phosphatase,β-galactosidase, glucose oxidase, G6-PDH and urease.
 8. The kit of claim4, wherein said reaction vessel means is a plurality of vessels, eachcontaining an equivalent amount of said monoclonal antibody.
 9. Animmunoassay for determining the presence of PCB's in a sample,comprising:(i) combining (ia) a monoclonal antibody with specificreactivity towards AROCLORs 1260, 1254, 1248, 1242, 1232, 1016 and 1221that is substantially the same as the monoclonal antibody produced byclone ATCC No. HB-12421, with (ib) a mixture of (ibi) the sample and(ibii) a hapten-and-reporter-molecule reagent which is cross reactivewith said monoclonal antibody, wherein said reporter molecule issusceptible to producing a detectable signal, to form an assay mixture;(ii) incubating said assay mixture to allow competitive monoclonalantibody binding between at least one of said AROCLORs, if present, inthe sample, and said reagent; (iii) causing production of saiddetectable signal and correlating said signal to the amount of reagentbound to said monoclonal antibody to obtain a measure of the amount ofPCB's in said sample.
 10. The immunoassay of claim 9, comprising usingan immobilized monoclonal antibody.
 11. The immunoassay of claim 9,comprising using said hapten-reporter molecule reagent of the formula##STR15## wherein X and Y independently represent a halogen n is aninteger from 0 to 5,m is an integer from 0 to 4, wherein n and m cannotboth be 0; R₅ is ##STR16## or a single bond R₆ is ##STR17## wherein R₇is hydrogen, C₁ -C₂ alkyls, linear, branched, or cyclic C₃ -C₆ alkyls;and p is O or an integer from 1 to 4; linked to a reporter molecule. 12.The immunoassay of claim 11, wherein said reporter molecule is an enzymeselected from the group consisting of horseradish peroxidase, alkalinephosphatases, β-galactosidase, glucose oxidase, G6-PDH and urease. 13.The immunoassay of claim 12, wherein said enzyme is horseradishperoxidase.
 14. The immunoassay of claim 9, further comprisingsimultaneously assaying a standard solution of at least one of AROCLOR1260, AROCLOR 1254 or AROCLOR 1248 and said sample.
 15. The kit of claim4, wherein said kit is a wipe test kit used to detect PCBs on a solidsurface and further comprises:(a) an extraction vessel having therein anextraction solvent; (b) a wiping means; and (c) a wiping template. 16.The kit of claim 15, wherein said wiping means is a cloth havingdimensions of 4 in by 4 in.
 17. The kit of claim 15, wherein saidextraction solvent is methanol.
 18. The kit of claim 15, wherein saidwiping template provides a controlled wiping area of 100 cm².
 19. Thekit of claim 4, wherein said kit is an immunoassay kit for testing ofsoil matrix samples and further comprises:(1) a dispersion vessel anddispersing means; (2) a dispersion solvent; and (2) a filtration means.20. The kit of claim 19, wherein said dispersing means is one or moreconventional dispersing pellets.
 21. The kit of claim 19, wherein saiddispersion solvent is methanol.
 22. The kit of claim 19, wherein saidfiltration means is a fingertip-operated filter unit fitted with one ormore non-adsorbing filters.
 23. A monoclonal antibody useful fordetecting the presence of PCB's in a sample, wherein said monoclonalantibody is a monoclonal antibody produced by clone ATCC No. HB-12421.24. An immunoassay kit useful for detecting the presence of PCB's in asample, comprising a monoclonal antibody produced by clone ATCC No.HB-12421.
 25. An immunoassay kit useful for detecting the presence ofPCB's in a sample, said kit comprising(i) a reaction vessel meanscontaining an immobilized monoclonal antibody wherein the immobilizedmonoclonal antibody is a monoclonal antibody produced by clone ATCC No.HB-12421, (ii) a standard solution comprising at least one of AROCLOR1260, AROCLOR 1254 or AROCLOR 1248, and a solvent; (iii) ahapten-reporter moiety-containing reagent which is cross-reactive withsaid antibody, and a suitable solvent; and (iv) means for causing saidreporter moiety to generate a signal and means for detecting the signal.26. An immunoassay for determining the presence of PCB's in a sample,comprising:(i) combining (ia) a monoclonal antibody produced by cloneATCC No. HB-12421, with (ib) a mixture of (ibi) the sample and (ibii) ahapten-and-reporter-molecule reagent which is cross reactive with saidmonoclonal antibody, wherein said reporter molecule is susceptible toproducing a detectable signal, to form an assay mixture; (ii) incubatingsaid assay mixture to allow competitive monoclonal antibody bindingbetween at least one of said AROCLORs, if present, in the sample, andsaid reagent; (iii) causing production of said detectable signal andcorrelating said signal to the amount of reagent bound to saidmonoclonal antibody to obtain a measure of the amount of PCB's in saidsample.